Cortical interneurons upregulate neurotrophins in vivo in response to targeted apoptotic degeneration of neighboring pyramidal neurons

The pressure-induced unfolding of wild-type staphylococcal nuclease (Snase WT) was studied using synchrotron X-ray small-angle scattering (SAXS) and Fourier-transform infrared (FT-IR) spectroscopy, which monitor changes in the tertiary and secondary structural properties of the protein upon pressurization. The experimental results reveal that application of high-pressure up to 3 kbar leads to an approximate twofold increase of the radius of gyration Rg of the native protein (Rg approximately 17 A) and a large broadening of the pair-distance-distribution function, indicating a transition from a globular to an ellipsoidal or extended chain structure. Analysis of the FT-IR amide I' spectral components reveals that the pressure-induced denaturation process sets in at 1.5 kbar at 25 degrees C and is accompanied by an increase in disordered and turn structures while the content of beta-sheets and alpha-helices drastically decreases. The pressure-induced denatured state above 3 kbar retains nonetheless some degree of beta-like secondary structure and the molecule cannot be described as a fully extended random coil. Temperature-induced denaturation involves a further unfolding of the protein molecule which is indicated by a larger Rg value and significantly lower fractional intensities of IR-bands associated with secondary-structure elements. In addition, we have carried out pressure-jump kinetics studies of the secondary-structural evolution and the degree of compactness in the folding/unfolding reactions of Snase. The effect of pressure on the kinetics arises from a larger positive activation volume for folding than for unfolding, and leads to a significant slowing down of the folding rate with increasing pressure. Moreover, the system becomes two-state under pressure. These properties make it ideal for probing multiple order parameters in order to compare the kinetics of changes in secondary structure by pressure-jump FT-IR and chain collapse by pressure-jump SAXS. After a pressure jump from 1 bar to 2.4 kbar at 20 degrees C, the radius of gyration increases in a first-order manner from 17 A to 22.4 A over a timescale of approximately 30 minutes. The increase in Rg value is caused by the formation of an extended (ellipsoidal) structure as indicated by the corresponding pair-distance-distribution function. Pressure-jump FT-IR studies reveal that the reversible first order changes in beta-sheet, alpha-helical and random structure occur on the same slow timescale as that observed for the scattering curves and for fluorescence. These studies indicate that the changes in secondary structure and chain compactness in the folding/unfolding reactions of Snase are probably dependent upon the same rate-limiting step as changes in tertiary structure.     

The folding kinetics and thermodynamics of the Fyn-SH3 domain

The equilibrium unfolding and the kinetic folding and unfolding of the 67 residue Fyn-SH3 domain have been investigated. Equilibrium unfolding experiments indicate that, despite the lack of both disulfide bonds and prosthetic groups, Fyn-SH3 is relatively stable with a free energy of folding of -6.0 +/- 0.6 kcal mol-1 at 20 degrees C. Kinetic experiments indicate that the domain refolds in a rapid two-state manner without significant population of intermediates (k = 94.3 s-1 in H2O at 20 degrees C). Despite the presence of two proline residues, the refolding of the domain is monophasic, and no significant proline isomerization-like refolding phase is observed. This can be attributed to an extremely low level of the incorrect (cis) isomer of the structurally important Pro134 residue in the protein denatured in 8 M guanidine hydrochloride. Analysis of the temperature and guanidine hydrochloride dependence of the folding rate suggests that the folding transition state of this protein is relatively well organized. A comparison with the refolding kinetics and thermodynamics of other homologous SH3 domains indicates that these exhibit an equivalent degree of transition state organization. This potentially arises from conservation of key features of the transition state conformation despite sometimes relatively low overall sequence identity. Such a comparison further suggests that relative thermodynamic stability is an important factor in determining the relative folding rates of natural proteins with a common fold, but that specific details of the amino acid sequence can also play a significant role in individual cases.     

Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4

One popular model for protein folding, the framework model, postulates initial formation of secondary structure elements, which then assemble into the native conformation. However, short peptides that correspond to secondary structure elements in proteins are often only marginally stable in isolation. A 33-residue peptide (GCN4-p1) corresponding to the GCN4 leucine zipper folds as a parallel, two-stranded coiled coil [O'Shea, E.K., Klemm, J.D., Kim, P.S., & Alber, T.A. (1991) Science 254, 539-544]. Deletion of the first residue (Arg 1) results in local, N-terminal unfolding of the coiled coil, suggesting that a stable subdomain of GCN4-p1 can form. N- and C-terminal deletion studies result in a 23-residue peptide, corresponding to residues 8-30 of GCN4-p1, that folds as a parallel, two-stranded coil with substantial stability (the melting temperature of a 1 mM solution is 43 degrees C at pH 7). In contrast, a closely related 23-residue peptide (residues 11-33 of GCN4-p1) is predominantly unfolded, even at 0 degrees C, as observed previously for many isolated peptides of similar length. Thus, specific tertiary packing interactions between two short units of secondary structure can be energetically more important in stabilizing folded structure than secondary structure propensities. These results provide strong support for the notion that stable, cooperatively folded subdomains are the important determinants of protein folding.     

Structure and stability of an early folding intermediate of Escherichia coli trp aporepressor measured by far-UV stopped-flow circular dichroism and 8-anilino-1-naphthalene sulfonate binding

The refolding kinetics of Escherichia coli trp aporepressor were monitored using stopped-flow far-ultraviolet circular dichroism and 8-anilino-1-naphthalene sulfonate fluorescence spectroscopy. Significant gains in secondary structure and the development of hydrophobic surface, respectively, were observed within the dead time of mixing (4-5 ms). These initial increases, or burst phase amplitudes, plotted as a function of final urea concentration, exhibited sigmoidal, coincident unfolding transition curves. The transition curves were fit to a two-state model, and the resulting free energies of folding in the absence of denaturant were found to be similar (approximately 3.3 kcal/mol). Three subsequent slow refolding phases exhibited relaxation times and amplitudes similar to those previously observed for tryptophan fluorescence [Gittelman, M. S., & Matthews, C. R. (1990) Biochemistry 29, 7011-7021]. These results support the proposals that a stable, monomeric intermediate is rapidly formed during the folding of trp aporepressor and that this species contains a significant amount of secondary structure and hydrophobic surface. This early intermediate is then processed through folding and association reactions that result in the formation of the remaining secondary, tertiary, and quaternary structure.     

The burst-phase intermediate in the refolding of beta-lactoglobulin studied by stopped-flow circular dichroism and absorption spectroscopy

The kinetics of the guanidine hydrochloride-induced unfolding and refolding of bovine beta-lactoglobulin, a predominantly beta-sheet protein in the native state, have been studied by stopped-flow circular dichroism and absorption measurements at pH 3.2 and 4.5 degrees C. The refolding reaction was a complex process composed of different kinetic phases, while the unfolding was a single-phase reaction. Most notably, a burst-phase intermediate of refolding, which was formed during the dead time of stopped-flow measurements (approximately 18 ms), showed more intense ellipticity signals in the peptide region below 240 nm than the native state, yielding overshoot behavior in the refolding curves. We have investigated the spectral properties and structural stability of the burst-phase intermediate and also the structural properties in the unfolded state in 4.0 M guanidine hydrochloride of the protein and its disulfide-cleaved derivative. The main conclusions are: (1) the more intense ellipticity of the intermediate in the peptide region arises from formation of non-native alpha-helical structure in the intermediate, apparently suggesting that the folding of beta-lactoglobulin is not represented by a simple sequential mechanism. (2) The burst-phase intermediate has, however, a number of properties in common with the folding intermediates or with the molten globule states of other globular proteins whose folding reactions are known to be represented by the sequential model. These properties include: the presence of the secondary structure without the specific tertiary structure; formation of a hydrophobic core; broad unfolding transition of the intermediate; and rapidity of formation of the intermediate. The burst-phase intermediate of beta-lactoglobulin is thus classified as the same species as the molten globule state. (3) The circular dichroism spectra of beta-lactoglobulin and its disulfide-cleaved derivative in 4.0 M guanidine hydrochloride suggests the presence of the residual beta-structure in the unfolded state and the stabilization of the beta-structure by disulfide bonds. Thus; if this residual beta-structure is part of the native beta-structure and forms a folding initiation site, the folding reaction of beta-lactoglobulin may not necessarily be inconsistent with the sequential model. The non-native alpha-helices in the burst-phase intermediate may be formed in an immature part of the protein molecule because of the local alpha-helical propensity in this part.     

Thermodynamic characterization of the coupled folding and association of heterodimeric coiled coils (leucine zippers)

Folding thermodynamics of nine heterodimeric, parallel coiled coils were studied by isothermal titration calorimetry (ITC) and thermal unfolding circular dichroism measurements. The heterodimers were composed of an acidic and a basic 30-residue peptide, which when in isolation were monomeric and essentially unstructured. The reaction followed a two-state mechanism indicating that folding and association were coupled. delta Hfold, delta Sfold and delta Cp normalized per mol of residue were of the same magnitude as for monomeric globular proteins, hence the energetics of folding and association of the heterodimeric coiled coils was balanced similarly to the folding of a single polypeptide chain. Cavity creating Leu/Ala substitutions revealed strong and position-dependent energetic coupling between leucine residues in the hydrophobic core of the coiled coil. delta Gunfold (equivalent to -delta Gfold in the two-state reaction) was determined from thermal unfolding. Global stability curves were calculated according to the Gibbs-Helmholtz equation and using the combined free energy data from ITC and thermal unfolding. Maximum stabilities were between 15 and 37 degrees C and cold denaturation could be demonstrated by direct calorimetry. The stability curves were based on free energies of folding measured between 10 and 85 degrees C and under identical solvent conditions. This represents a novel experimental approach which circumvents the use of varying solvent conditions as is typically required to measure protein stability curves. Discrepancies were noticed between van't Hoff enthalpies deduced from thermal unfolding and measured by direct calorimetry. The discrepancies are thought to be due to residual ordered structure in the denatured single chains around room temperature but not near the transition midpoint temperature Tm. This demonstrates that over an extended temperature range the assumption of a common denatured state implicit in the van't Hoff analysis may not always be valid.     

Reduced-denatured ribonuclease A is not in a compact state

Dynamic light scattering and circular dichroism experiments were performed to determine the compactness and residual secondary structure of reduced and by 6 M guanidine hydrochloride denatured ribonuclease A. We find that reduction of the four disulphide bonds by dithiothreitol at 20 degrees C leads to total unfolding and that a temperature increase has no further effect on the dimension. The Stokes' radius of ribonuclease A at 20 degrees C is R(s) = (1.90 +/- 0.04) nm (native) and R(s) = (3.14 +/- 0.06) nm (reduced-denatured). Furthermore, circular dichroism spectra do not indicate any residual secondary structure. We suggest that reduced-denatured Ribonuclease A has a random coil-like conformation and is not in a compact denatured state.     

Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability

Folding stabilities of camelized human antibody VH domains were studied through the determination of their melting points in thermodenaturation experiments. The melting point of a VH domain originating from a synthetic library of human VHs, which had been optimized for the use as small recognition units through the mimicking of camelid antibody heavy chains occurring naturally without light chain, was 56.6 degrees C compared with 71.2 degrees C of the original human VH. Its stability was improved (melting point 61.6 degrees C) through three mutations to mimic camelid VHs even further: Va137 was replaced by phenylalanine and two cysteines were introduced at position 33 and 100b. The resulting VH folded properly and formed a second intradomain disulphide between the extra cysteines. The new mutations were then built constitutively into a phage-display VH library, from which antigen-specific VHs were selected. Two were analysed for stability with melting points of 72.6 and 75.3 degrees C. Thus secondary camelization enabled the isolation of VHs with improved folding stabilities exceeding even that of the original human VH. This indicates an effect on folding stability for some mutations specific in the light chain lacking camelid heavy chains.     

Denaturation and association of DNA sequences by certain polypropylene surfaces

UDP-galactose 4-epimerase from yeast Kluyveromyces fragilis is a dimeric molecule of 75 kDa per subunit with one molecule of cofactor NAD per dimer. It undergoes unfolding and complete dissociation in presence of 8 M urea at pH 7.0 by 10 min. It can be functionally reconstituted almost quantitatively in 2 h by dilution with 20 mM sodium phosphate buffer, pH 7 containing 1 mM extraneous NAD under a second order kinetics [Bhattacharyya, D. (1993) Biochemistry 32, 9726-9734]. Denaturation between 10-60 min inversely affects both the rate and maximum recovery of activity upon refolding. Aggregation of this protein has not been observed under these conditions. The time dependent reaction at the unfolded state is independent of pH between 5.4-10.4 but strongly dependent on temperature of denaturation between 0-20 degrees C. Unfolding at 0 degrees C divides the protein largely into two populations-34% of fast folding species following an apparent first order kinetics and 59% of slow folding species following a second order kinetics of reactivation. A very fast folding species of low abundance 3.5-7.5% depending on temperature of denaturation has been identified, which gets active status within the dead time of mixing. Interaction with the active site directed fluorescence probe 1-anilino 8-naphthalene sulfonic acid (1-ANS) and estimation of bound NAD suggest that the catalytic region of this enzyme is not formed in the long term denatured samples. The whole process of reactivation is catalysed by peptidyl prolyl cis-trans isomerase and thus suggests that one or more proline residues stereochemically control the rate limiting step of reactivation.     

The analgesic effects of tryptophan and its metabolites in the rat

Structural evolution during the phase transition from h (hexagonal)- to c (cubic)-boron nitrides (BN) under high pressure (6.5-7.7 GPa) at high temperature (1,700-2,150 degrees C) was examined by using high-resolution transmission electron microscopy (HRTEM) and electron energy loss spectroscopy (EELS). At the initial stage of the evolution, some starting h-BN plates were strongly folded, while others were slightly bent. As a result, a strong texture was formed. HRTEM revealed that the interplanar distance between sp2 sheets became slightly shortened and they were slightly sheared to each other during the folding and bending. As a result, m (monoclinic)-BN was formed near the folding plane with lattice parameters; a = 0.433 nm, b = 0.250 nm, c = 0.32-0.33 nm, and beta = 90-92 degrees. In a succeeding stage, the value of beta increased to 92-95 degrees. c-BN grains appeared with nano-scale twins and sometimes partly included wurtzite-type BN. They started to grow with secondary twins at higher temperature. EELS analysis revealed that the band structure of sp2 sheets changed during the transition from h-BN to m-BN; the density of state for the pi* bond became prominently high in m-BN as compared to that in h-BN.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Conformational stability of factor VIIa: biophysical studies of thermal and guanidine hydrochloride-induced denaturation

The binding of the multidomain protein factor VIIa (fVIIa) to tissue factor provides the interprotein communication necessary to make fVIIa an efficient catalyst of the initial event in the extrinsic pathway of blood coagulation. We have investigated the stability of individual domains in fVIIa and the influence of Ca2+ and an irreversible active-site inhibitor (FFR-chloromethyl ketone). Equilibrium guanidine hydrochloride (GuHCl)-induced unfolding monitored by tryptophan fluorescence and far-UV circular dichroism (CD) demonstrated that the gamma-carboxyglutamic acid (Gla) domain unfolds at 0.3 M GuHCl and the serine protease (SP) domain at 3 M GuHCl and that Ca2+ is a prerequisite for the formation of an ordered, compact structure in the Gla domain. The loss of amidolytic activity coincides with the first transition, which is stabilized by the active-site inhibitor, and a change in the environment of the active site is demonstrated using a fluorescent inhibitor (DEGR-chloromethyl ketone). Thermal unfolding monitored by differential scanning calorimetry (DSC) reveals that Ca2+ stabilizes the SP domain slightly, increasing the unfolding temperature by 2.7 degrees C. In addition, Ca2+ is required for a large enthalpy change concomitant with unfolding of the Gla domain, and this unfolding enthalpy is only detectable in the presence of the SP domain, indicating some kind of interaction between these domains. Thermal unfolding measured by CD indicates secondary structural changes at the same temperature as the heat absorption in the DSC but only when both the Gla domain and the SP domain are present together with Ca2+ ions. Taken together, these results indicate a Ca2+-dependent interaction between the Gla domain and the SP domain, implying a high degree of flexibility of the domains in free fVIIa. It is also shown that the epidermal growth factor-like domains are stable at elevated temperatures and high GuHCl concentrations. Moreover, already at physiological temperature, subtle structural changes take place which influence the overall shape of fVIIa and are detrimental to its enzymatic activity.     

Folding mechanism of the alpha-subunit of tryptophan synthase, an alpha/beta barrel protein: global analysis highlights the interconversion of multiple native, intermediate, and unfolded forms through parallel channels

A variety of techniques have been used to investigate the urea-induced kinetic folding mechanism of the alpha-subunit of tryptophan synthase from Escherichia coli. A distinctive property of this 29 kDa alpha/beta barrel protein is the presence of two stable equilibrium intermediates, populated at approximately 3 and 5 M urea. The refolding process displays multiple kinetic phases whose lifetimes span the submillisecond to greater than 100 s time scale; unfolding studies yield two relaxation times on the order of 10-100 s. In an effort to understand the populations and structural properties of both the stable and transient intermediates, stopped-flow, manual-mixing, and equilibrium circular dichroism data were globally fit to various kinetic models. Refolding and unfolding experiments from various initial urea concentrations as well as forward and reverse double-jump experiments were critical for model discrimination. The simplest kinetic model that is consistent with all of the available data involves four slowly interconverting unfolded forms that collapse within 5 ms to a marginally stable intermediate with significant secondary structure. This early intermediate is an off-pathway species that must unfold to populate a set of four on-pathway intermediates that correspond to the 3 M urea equilibrium intermediate. Reequilibrations among these conformers act as rate-limiting steps in folding for a majority of the population. A fraction of the native conformation appears in less than 1 s at 25 degrees C, demonstrating that even large proteins can rapidly traverse a complex energy surface.     

Is the continuity of the domains required for the correct folding of a two-domain protein?

The role of domains in protein folding has been widely studied and discussed. Nevertheless, it is not clear whether the continuity of the domains in a protein is an essential requirement in determining the folding pathway. Previous studies have shown that the isolated structural domains of the two-domain monomeric enzyme, yeast phosphoglycerate kinase (yPGK), are able to fold independently into a quasinative structure, but they neither reassociate nor generate a functional enzyme [Minard, P., Hall, L., Betton, J. M., Missiakas, D., & Yon, J. M. (1989) Protein Eng. 3, 55-60; Fairbrother, W. J., Bowen, D., Hall, L., Williams, R. J. P. (1989) Eur. J. Biochem. 184, 617-625; Missiakas, D., Betton, J. M., Minard, P., & Yon, J. M. (1990) Biochemistry 29, 8683-8689]. In the present work, two circularly permuted variants of the yPGK gene were constructed. The natural adjacent chain termini were directly connected and the new extremities were created within the N-domain (at residues 71 and 72) or the C-domain (at residues 291 and 292), respectively. These two proteins were overexpressed and purified. They exhibit 14% and 23% of the wild-type enzyme activity, respectively. The two mutants fold in a compact conformation with slight changes in the secondary and tertiary structure probably related to the presence of a heterogeneous population of molecules. The unfolding studies reveal a large decrease in stability. From the present data it appears that, although the circular permutations induce some perturbations in the structure and stability of the protein, the continuity of the domains is not required for the protein to reach a native-like and functional structure.     

The equilibrium intermediate of beta-lactoglobulin with non-native alpha-helical structure

It is generally considered that intermediates of protein folding contain partially formed native-like secondary structures. In contrast, we recently reported that the kinetic folding intermediate of bovine beta-lactoglobulin contains non-native alpha-helical structures. To understand the mechanism that stabilizes the non-native intermediate, we characterized by circular dichroism (CD) the equilibrium unfolding transition of beta-lactoglobulin induced by guanidine hydrochloride (Gdn-HCl) at pH 2 and 4 degrees C. The unfolding transition measured by near-UV CD preceded the transition measured by far-UV CD, indicating the accumulation of the intermediate state. The far-UV CD spectrum of the intermediate, obtained by global fitting analysis of the CD spectra in the presence of various concentrations of Gdn-HCl, was similar to the burst-phase intermediate observed in the refolding kinetics, and contained non-native alpha-helical structures. Addition of 10% (v/v) 2,2,2-trifluoroethanol (TFE) increased the helical content of the equilibrium intermediate, although the protein still assumed the native structure in the absence of Gdn-HCl. A phase diagram of the conformational states, i.e. the alpha-helical intermediate, unfolded and native states, against the concentration of TFE and Gdn-HCl was constructed. This indicated that, because of the high helical preference of the amino acid sequence of beta-lactoglobulin, the helical region protrudes into the boundary between the native and unfolded states, resulting in non-monotonic accumulation of the helical intermediate upon equilibrium unfolding of the native beta-sheet structure. This is the first observation to indicate that a non-native alpha-helical intermediate accumulates during equilibrium unfolding of a predominantly beta-sheet protein.     

p53 protein accumulation and response to adjuvant chemotherapy in premenopausal women with node-negative early breast cancer

Although beta-sheets represent a sizable fraction of the secondary structure found in proteins, the forces guiding the formation of beta-sheets are still not well understood. Here we examine the folding of a small, all beta-sheet protein, the E. coli major cold shock protein CspA, using both equilibrium and kinetic methods. The equilibrium denaturation of CspA is reversible and displays a single transition between folded and unfolded states. The kinetic traces of the unfolding and refolding of CspA studied by stopped-flow fluorescence spectroscopy are monoexponential and thus also consistent with a two-state model. In the absence of denaturant, CspA refolds very fast with a time constant of 5 ms. The unfolding of CspA is also rapid, and at urea concentrations above the denaturation midpoint, the rate of unfolding is largely independent of urea concentration. This suggests that the transition state ensemble more closely resembles the native state in terms of solvent accessibility than the denatured state. Based on the model of a compact transition state and on an unusual structural feature of CspA, a solvent-exposed cluster of aromatic side chains, we propose a novel folding mechanism for CspA. We have also investigated the possible complications that may arise from attaching polyhistidine affinity tags to the carboxy and amino termini of CspA.     

Extremely fast folding of a very stable leucine zipper with a strengthened hydrophobic core and lacking electrostatic interactions between helices

The dimer interface of a leucine zipper involves hydrophobic as well as electrostatic interactions between the component helices. Here we ask how hydrophobic effects and electrostatic repulsion balance the rate of folding and thermodynamic stability of a designed dimeric leucine zipper formed by the acidic peptide A that contains four repeating sequence units, (abcdefg)4. The aliphatic a and d residues of peptide A were the same as in the GCN4 leucine zipper but the e and g positions were occupied by Glu, which prevented folding above pH 6 because of electrostatic repulsion. Leucine zipper A2 was formed by protonation of the e and g side chains with a sharp transition midpoint at pH 5.2. Folding could be described by a two-state transition from two unfolded random coil monomers to a coiled coil dimer. There was a linear relationship between the logarithm of the rate constants and the number of repulsive charges on the folded leucine zipper dimer. The same linear relationship applied to the free energy of unfolding and the number of repulsive charges at thermodynamic equilibrium. Fully protonated peptide A folded at a near diffusion-limited rate (kon = 3 x 10(8) M-1 s-1), and the free energy of folding was -55 kJ mol-1 at 25 degrees C. The present work shows that protonation of Glu in positions e and g increases both the folding rate and the stability of the leucine zipper in the absence of any interhelical electrostatic interactions. Protonated Glu is proposed to act like a nonpolar residue and to strengthen the hydrophobic core by folding back toward the core residues in the a and d positions. This effect adds more to the free energy of unfolding and to the rate of folding than maximizing the number of salt bridges across the helix interface in an electrostatically stabilized heterodimeric leucine zipper [Wendt, H., Leder, L., H?rm?, H., Jelesarov, I., Baici, A., and Bosshard, H. R. (1997) Biochemistry 36, 204-213].     

Nature of the unfolded state of ribonuclease A: effect of cis-trans X-Pro peptide bond isomerization

The equilibrium unfolded state of disulfide-intact bovine pancreatic ribonuclease A is a heterogeneous mixture of unfolded species. Previously, four unfolded species have been detected experimentally. They are Uvf, Uf, UsII, and UsI which have refolding time constants on the millisecond, millisecond to second, second to tens of seconds, and hundreds of seconds time scales, respectively. In the current study, the refolding pathway of the protein was investigated under favorable folding conditions of 0.58 M GdnHCl, pH 5.0, and 15 degrees C. In addition to the above four unfolded species, the presence of a fifth unfolded species was detected. It has a refolding time constant on the order of 2 s under the conditions employed. This new unfolded species is labeled Um, for medium-refolding species. Single-jump refolding experiments monitored by tyrosine burial and by cytidine 2'-monophosphate inhibitor binding indicate that the different unfolded species refold to the native state along independent refolding pathways. The buildup of the different unfolded species upon unfolding of the protein from the native state was monitored by absorbance using double-jump experiments. These experiments were carried out at 15 degrees C and consisted of an unfolding step at 4.2 M GdnHCl and pH 2.0, followed, after a variable delay time, by a refolding step at 0.58 M GdnHCl and pH 5.0. The results of these experiments support the conclusion that the different unfolded species arise from cis-trans isomerizations at the X-Pro peptide bonds of Pro 93, 114, and 117 in the unfolded state of the protein. The rates of these isomerizations were obtained for each of these three X-Pro peptide bonds at 15 degrees C.     

Slow alpha helix formation during folding of a membrane protein

Very little is known about the folding of proteins within biological membranes. A "two-stage" model has been proposed on thermodynamic grounds for the folding of alpha helical, integral membrane proteins, the first stage of which involves formation of transmembrane alpha helices that are proposed to behave as autonomous folding domains. Here, we investigate alpha helix formation in bacteriorhodopsin and present a time-resolved circular dichroism study of the slow in vitro folding of this protein. We show that, although some of the protein's alpha helices form early, a significant part of the protein's secondary structure appears to form late in the folding process. Over 30 amino acids, equivalent to at least one of bacteriorhodopsin's seven transmembrane segments, slowly fold from disordered structures to alpha helices with an apparent rate constant of about 0.012 s-1 at pH 6 or 0.0077 s-1 at pH 8. This is a rate-limiting step in protein folding, which is dependent on the pH and the composition of the lipid bilayer.     

Recognition between disordered states: kinetics of the self-assembly of thioredoxin fragments

The disordered N- (1-73) and C- (74-108) fragments of oxidized Escherichia colithioredoxin (Trx) reconstitute the native structure upon association [Tasayco, M. L., & Chao, K. (1995) Proteins: Struct., Funct., Genet. 22, 41-44]. Kinetic measurements of the formation of the complex (1-73/74-108) at 20 degrees C under apparent pseudo-first-order conditions using stopped-flow far-UV CD and fluorescence spectroscopies indicate association coupled to folding, an apparent rate constant of association [kon = (1330 +/- 54) M-1 s-1], and two apparent unimolecular rate constants [k1 = (0. 037 +/- 0.007) s-1 and k2 = (0.0020 +/- 0.0005) s-1]. The refolding kinetics of the GuHCl denatured Trx shows the same two slowest rate constants. An excess of N- over C-fragment decreases the kon, and the slowest phase disappears when a P76A variant is used. Stopped-flow fluorescence measurements at 20 degrees C indicate a GuHCl-dependent biphasic dissociation/unfolding process of the complex, where the slowest phase corresponds to 90% of the total. Their rate constants, extrapolated to zero denaturant, k-1 = (9 +/- 3) x 10(-5) s-1 and k-2 = (3.4 +/- 1.2) x 10(-5) s-1, show m# values of (4.0 +/- 0.4) kcal mol-1 M-1 and (3.5 +/- 0.1) kcal mol-1 M-1, respectively. Our results indicate that: (i) a compact intermediate with trans P76 and defined tertiary structure seems to participate in both the folding and unfolding processes; (ii) not all the N-fragment is competent to associate with the C-fragment; (iii) conversion to an association competent form occurs apparently on the time scale of P76 isomerization; and (iv) the P76A variation does not alter the association competency of the C-fragment, but it permits its association with "noncompetent" forms of the N-fragment.